1. Field of the Invention
This invention relates generally to improved cyanine and indocyanine bioconjugates; and, particularly to improved site-specific delivery for optical tomographic, endoscopic, photoacoustic, sonofluorescent, laser assisted guided surgery, and therapeutic purposes.
2. Background of the Prior Art
Several dyes, including derivatives of fluorescein and carbocyanine, that emit light in the visible and near-infrared region of the electromagnetic spectrum have in the past and are currently being used for various biomedical applications due to their biocompatibility, high molar absorptivity, or high fluorescence quantum yields. This high sensitivity parallels that of nuclear medicine and permits visualization of organs and tissues without the negative effect of ionizing radiation. Most dyes lack specificity for particular organs or tissues and, hence, these dyes must be attached to bioactive carriers such as proteins, peptides, carbohydrates, and the like to deliver the dyes to specific regions in the body. Several studies on the use of near infrared dyes and dye-biomolecule conjugates have been published (Patonay et al., 1991; Slavik, 1994 Brinkley, 1993; Lee and Woo, U.S. Pat. No. 5,453,505; Hohenschuh, WO 98/48846; Turner et al., WO 98/22146; Licha et al., WO 96/17628; and Snow et al., WO 98/48838). Of particular interest is the targeting of tumor cells with antibodies or other large protein carriers as delivery vehicles (Becker, et al., 1999). Such an approach has been widely used in nuclear medical applications, and the major advantage is the retention of a carrier's tissue specificity since the molecular volume of the dye is substantially smaller than the carrier. However, this approach does have some serious limitations in that the diffusion of high molecular weight bioconjugates to tumor cells is highly unfavorable, and is further complicated by the net positive pressure in solid tumors (Jain, 1994). Furthermore, many dyes in general, and cyanine dyes, in particular, tend to form aggregates in aqueous media that lead to fluorescence quenching.
Therefore, to solve these problems, U.S. Pat. No. 6,217,848 disclosed dye-peptide conjugates, including several cyanine dyes with a variety of bis- and tetrakis (carboxylic acid) homologues. The small size of the compounds allowed more favorable delivery to tumor cells, as compared to larger molecular weight imaging agents.
A typical example of the cyanine compounds used to make these conjugates is indoacyanine green (ICG) which absorbs and emits light in the near infrared region (NIR) wavelengths.
However, many drawbacks have been encountered in the use of these recently developed compounds. First, it is difficult to alter their spectral properties to coincide with a desired biological or chemical event, thereby limiting the scope of their functionality as imaging agents. For example, the fluorescence emission and fluorescence lifetime of ICG, in the tissue itself, do not significantly change in situ; and hence, the ICG derivatives cannot be used effectively as a reporter molecules to monitor the functional events such as enzyme activity, and gene expression, as they occur. To circumvent this problem, recent studies have used a “pro-drug” approach, where the fluorescence signal, from a pre-quenched carbocyanine compound, is detected in response to a diagnostic biological event, such as increased local acidity in solid tumors, or high expression of some proteases in metastatic tumors [Bremer C., et al,. “Imaging of differential protease expression in breast cancers for detection of aggressive tumor phenotypes”, Radiology 222, 814-818 (2002); Weissleder R., “A clearer vision for in vivo imaging”, Nat. Biotechnol. 19, 316-317 (2001)].
Unfortunately, this “pro-drug” approach relies on the stacking of dyes on a polymer backbone to achieve some level of decrease in fluorescence emission. In addition, the large size of the copolymer-probe conjugate used precludes rapid delivery of the probe to solid tumors. Moreover, the delivery method is nonspecific and the major photophysical feature of the dye that was affected was a reduction in the fluorescence emission of the polymeric material, rather than the tissue itself. Thus, availability of intramolecularly-quenched carbocyanine compounds, coupled with specific delivery to target tissue for functional imaging events such as enzyme activity and gene expression remain an unmet need.
Another problem with the recent carbocyanine dye-bioconjugates, is in vivo instability. That is, the bioactive carrier molecule, such as a peptide, is attached to the dye via its N-terminus, where the peptide is susceptible to degradation by exopeptidases via the C-terminus.
Also, the routine use of cyanine bioconjugate compounds in clinical settings as imaging agents is inhibited by the potential for hepatobilliary toxicity resulting from the rapid clearance of these dyes by the liver. This is associated with the tendency of carbocyanine compounds to form aggregates in solution, which could be taken up by Kupffer cells in the liver. Various attempts to obviate this problem have not been very successful. Typically, hydrophilic peptides, polyethyleneglycol or oligosaccharide conjugates have been used but these resulted in excessively long-circulating products that are eventually cleared by the liver.
It would be a welcomed advancement in the industry to overcome the aforesaid drawbacks of the prior art.
The publications and other materials used herein to support the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are respectively listed in the appended List of Reference.